The present invention relates to a method and apparatus for screening for serum apolipoproteins as a biochemical indicator of coronary risk.
The association between elevated levels of serum cholesterol and heart disease has become one of the most cognizant risk factors in adult populations. Recent studies have shown that one in five adults die of coronary heart disease as a direct result of elevated serum cholesterol levels, and one in three is at risk of the disease. However, early screening of patients for this health risk is not routinely performed by physicians unless specifically requested by the patient. This is due in part to reliance on the patient's family health history as an indicator of negative risk factor in cardiovascular disease and the expense and inconvenience of the testing procedure. Often the family physician does not have a convenient test apparatus that can be routinely used in the office for the determination of cholesterol and is therefore dependent on an analysis by an outside clinical laboratory to provide the data. In addition, variations exist in the screening procedures currently used by clinical laboratories to determine serum cholesterol and many procedures are expensive to the patient, time-consuming, and are prone to large variability which may lead to errors in diagnosis and treatment.
Numerous investigations have demonstrated an inverse correlation between heart disease and plasma levels of high density lipoprotein (HDL), a carrier (vehicle) of cholesterol. Specifically, elevated levels of HDL cholesterol have been associated with lower incidence of heart disease. Other vehicles of cholesterol transport are defined in terms of their hydrated densities. These include very low density lipoprotein (VLDL) d&lt;1.006 g/ml, low density lipoprotein (LDL) d=1.006-1.063 g/ml, and HDL d=1.063-1.21/g/ml.
One procedure commonly used to determine HDL cholesterol involves the precipitation of VLDL and LDL followed by centrifugation. The supernatant, which contains HDL-associated cholesterol, is then chemically or enzymatically assayed for cholesterol.
Several clinical studies suggest that direct measurement of LDL precipitate is a better indicator of the risk for coronary heart disease than nonspecific measurement of HDL cholesterol. In one procedure known in the art, VLDL and LDL are precipitated from serum, the mixture is centrifuged, and the volume of the precipitate is measured. Lehmann et al, Lab. Med., 14:782, (1983). This volume can be directly related to the concentrations of VLDL and LDL. This procedure also enables one to determine the concentration of total cholesterol plus triglycerides as a screening test for hyperlipidemia. However, this method of predicting heart disease or the predisposition therefore is too imprecise for this procedure to serve as a mass screening program for large numbers of subject children and adults. This procedure does not predict the sum of cholesterol and triglyceride in a linear fashion. In addition, this procedure has been only performed on blood samples obtained by venous puncture because of the large blood volume required. Utilization of blood from a finger-stick source does not provide a sufficient volume of blood necessary to perform currently available analyses.
While far less blood can be obtained from finger-stick sampling of an individual than can be obtained from venous sampling, experience indicates that finger-stick blood sampling is much better tolerated and accepted by a subject than is venous sampling. Thus, the ability to use blood from finger-stick samples for assays of blood components increases the participation rate of subjects, and particularly children, in mass screening programs.
Further, it is known that some individuals with coronary heart disease have normal cholesterol levels. This fact must be kept in mind when developing a method and device for screening large numbers of people. A possible explanation is that these "normocholesterolemic" individuals have abnormalities in cholesterol metabolism not detected by measuring the total cholesterol level.
The importance of apolipoproteins and the increased interest in the measurement of these proteins stems from the specific function each class of apolipoprotein has in the mechanism of lipoprotein metabolism and their relationship to a diseased state. In LDL metabolism, cholesterol is delivered to peripheral cells by LDL receptor sites located on the cell membrane. Elevated levels of intracellular cholesterol cause suppression of LDL receptor activity and subsequent accumulation of atherogenic LDL. Apo-B is the structural entity of LDL that is responsible for LDL receptor recognition by the peripheral cells. Whereas LDL particles are heterogeneous in terms of size and composition, each LDL particle has one molecule of Apo-B per particle. Thus, atherogenicity is best measured by the Apo-B component rather than by the varying number of cholesterol components.
Hyperapo-B-lipoproteinemia, detectable even in the pediatric age group, as well as in adults, is very prevalent in patients with coronary artery disease. In men, low-density lipoprotein cholesterol and apolipoprotein B levels correlated best with severity of the disease; in women the best discriminators were LDL and intermediate density lipoprotein (IDL) triglycerides, LDL cholesterol, and LDL/apolipoprotein B.
In hyperapolipoprotein B there is an increased number of atherogenic LDL particles in the blood that have an altered chemical composition: the core is depleted of cholesterol ester, and relatively enriched with apolipoprotein B. Increased numbers of smaller denser LDL particles result. Tests which measure only total cholesterol or LDL cholesterol underestimate the number of atherogenic LDL particles. In some families increased levels of apolipoprotein B may indicate the presence of lipoprotein phenotypes IV, IIa, and IIB, indicative of familial combined hyperlipidemia, that is so prevalent in survivors of myocardial infarctions.
Using cholesterol levels alone to screen asymptomatic adults for risk of coronary artery disease will give some individuals false assurance that they are at a low risk for a heart attack if these individuals have a normal cholesterol and elevated levels of LDL, low levels of HDL, or elevated levels of apolipoprotein B. Primarily due to cost, it is not usually recommended that mass screening programs measure triglycerides, HDL cholesterol, or LDL cholesterol.
Correlation of Apo-B blood levels and the severity of clinically verified coronary artery disease in patients has been evaluated by Naito. Ann. NY Acad. Sci. 454:230-238, (1985). Subjects of this study were divided into four categories: 1) asymptomatic patients not undergoing coronary angiography; 2) those with clinically nonsignificant coronary artery disease; 3) those with clinically significant coronary artery disease; and 4) those with severe coronary artery disease. Clinical verification of diseased states in each category was accomplished by coronary angiography. The presence or absence of coronary artery disease was assessed on the basis of the degree of stenosis within any segment of the arterial tree. Serum Apo-B levels were determined for ten subjects in each category by an immunochemical assay and the data obtained therefrom are set forth in Table 1.
TABLE 1 ______________________________________ Relation of Apo-B Concentrations (mg/dl) to the Progression of Coronary Heart Disease (CHD). Normal No Significant Severe Asymptomatic Significant CHD.sup.b CHD.sup.c Subjects CHD.sup.a (N = 10) (N = 10) (N = 10) (N = 10) Mean + Mean + Mean + SD Mean + SD SD SD ______________________________________ Apo B 107 .+-. 25 116 .+-. 24 129 .+-. 20 137 .+-. 26 ______________________________________ .sup.a 1-50% stenosis in at least one coronary arterial segment. .sup.b 51-99% stenosis in at least one coronary arterial segment. .sup.c 100% stenosis in at least one coronary arterial segment.
It is evident that in order to avoid false negative reports, effective screening of children and adults for the development of premature heart disease must include the measurement of apolipoprotein B. However, due to the high cost of currently available methods for measurement of apolipoprotein B, they are impractical for use as mass screening tests.
What was needed was a rapid, inexpensive, accurate and reliable method to predict individuals with high coronary risk. What was also needed was an effective method for predicting coronary risk in an individual which could be used in mass screening tests.